Glutathione S-transferase (GST) expression was examined in hepatic cytosol from rats and rabbits treated with 4-picoline, pyrrole, pyridine, pyrazine, imidazole, or piperidine using enzymatic activity, SDS-PAGE, and immunoblot analyses and the results were compared to those obtained with phenobarbital and 3-methylcholanthrene. SDS-PAGE and immunoblot analyses of hepatic cytosol prepared from rats treated with pyrazine revealed the induction of class alpha (Ya and Yc) and mu (Yb) bands with a corresponding 2.4-fold increase in metabolic activity using 1-chloro-2,4-dinitrobenzene as substrate. A new class alpha band migrating in the region of the Yc band was observed in the SDS-PAGE and detected in the immunoblot of cytosol from pyrrole-treated rats, whereas treatment with 4-picoline, imidazole, or piperidine failed to alter the expression of the major classes of GST isozymes in this species. SDS-PAGE and immunoblot analyses of rabbit hepatic cytosol revealed a unique species-dependent difference in the expression of GSTs. While phenobarbital and 3-methylcholanthrene induce class alpha and mu GST expression in rat hepatic cytosol, one of the most interesting observations was that neither of these agents stimulated GST expression in the rabbit. Immunoblot analysis of cytosol isolated from 4-picoline-treated rabbits using GST class alpha-specific IgG showed the appearance of a novel class alpha 28-kDa GST band and the concomitant disappearance of a class alpha 29-kDa GST band. In addition, SDS-PAGE and immunoblot analyses showed that treatment of rabbits with pyrrole, pyrazine, imidazole, or piperidine resulted in the disappearance of this class alpha 29-kDa GST band with no detectable expression of the class alpha 28-kDa GST band; the level of the class alpha 29-kDa band was unaffected by pyridine treatment. In contrast, immunoblot analyses of hepatic cytosol revealed that a 25.5-kDa class mu GST band disappeared following treatment with pyridine, but was unaffected by treatment with other nitrogen heterocycles. The Vmax of glutathione conjugation to the substrate 1-chloro-2,4-dinitrobenzene decreased by 52, 36, 59, 41, 37, and 32% in hepatic cytosol isolated from 4-picoline-, pyrrole-, pyridine-, pyrazine-, imidazole-, and piperidine-treated rabbits, respectively. The results suggest that nitrogen heterocycles differ in their ability to modulate glutathione S-transferase isozyme expression in rat and rabbit hepatic tissue and that rabbit hepatic GSTs are refractory to induction by agents such as pyrazine, phenobarbital, or 3-methylcholanthrene and hence these xenobiotics do not appear to be bifunctional inducers in this species.